Los autores desean agradecer a José LoTurco y Dennis Selkoe útil para los debates sobre esta técnica. Los autores agradecen a los donantes de la Fundación de Asistencia para la Salud de América, para el apoyo de esta investigación.

El protocolo básico para la electroporación en el útero ha sido descrito en detalle en otro artículo Jove de laboratorio Kriegstein 1, 2. Esta técnica fue descrita originalmente en el 3 Osumi de laboratorio y el protocolo se basa en la desarrollada en el laboratorio LoTurco 4. Vamos a ofrecer una visión general del nuestro protocolo de electroporación en el útero de embriones de rata, centrándose en los detalles más importantes, seguido de una descripción de nuestro protocolo que se aplica en la electroporación in utero al estudio de la función de genes en consecuencia el proceso neuronal. 1. En electroporación in utero <ol><li> Preparación de las agujas del ADN y de carga El primer paso en el electroporations útero es el diseño de su experimento para determinar qué construcciones de ADN que desee inyectar. Este método es útil tanto para misexpressing o derribar los genes de interés. Si usted está planeando en misexpressing o sobreexpresión de un gen, asegúrese de usar un promotor que se activa en las células precursoras neuronales. Recomendamos el promotor CAGGS, que consiste en el pollo beta-actina promotor y potenciador de CMV 5. Ya que sólo un pequeño subconjunto de las células se transfectaron usando electroporación en el útero, es importante incluir un plásmido que codifica una proteína fluorescente GFP, como para que pueda seguir las células que se electroporated con éxito. Para el plásmido que codifica GFP, se recomienda preparar el ADN a una concentración de 0,5 microgramos por microlitro. Para las construcciones shRNA, hemos encontrado que 0,5-1,0 mg por l de resultados eficientes derribar de su gen de interés. Para la sobreexpresión o misexpression, se utiliza entre 1,0 y 3,0 microgramos por microlitro, dependiendo del tamaño del gen y el nivel de expresión de que el experimento requiere. ADN se preparan utilizando una Qiagen libre de endotoxinas kit de preparación, y se diluye en 1 x PBS. Se inyecta aproximadamente 0,5-1,0 l por el cerebro embrionario, por lo que, por una camada de animales que preparar 10 l de mezcla de ADN para la inyección. Añadimos una L de Fast Green en el ADN para que podamos seguir el ADN inyectado. Tirar las agujas de la forma correcta es un paso crítico. Walantus et al. Utiliza un sistema diferente para la entrega de los ADN y por lo que su preparación para la aguja es 1,2 entonces nuestra ligeramente diferente. La configuración que se utiliza para tirar de las agujas dependerá de la marca de la aguja de extractor que usted tiene. Utilizamos el modelo 750 de David Kopf. La configuración que utilizamos son: Heat 1: 9,0, Heat 2: 0, Soleniod: 0, el tamaño de filamento de 3,0 mm, proximidad del calentador: 3 mm, Tiempo: 10 seg. Una vez que sacó, nos cortamos las agujas con una hoja de afeitar en un ángulo de ~ 45 grados de tal manera que la distancia de la mayor parte de la apertura a la punta es de 11 mm. A continuación, cargar el ADN de la parte de atrás de la aguja. A continuación, llenar el espacio que queda en la aguja con el aceite de maíz. Para la inyección de ADN, se utiliza un Picospritzer III. Dependiendo del bisel exacto en que se corta para cada aguja, se establece el Picospritzer 4,0 a 6,0. Nosotros usamos un pedal de pie para entregar el aire comprimido que expulsa el ADN de la aguja. </li><li> La preparación de animales para la cirugía Usamos libres de patógenos ratas Sprague Dawley, exclusivamente para estas operaciones. Varios otros laboratorios utilizan ratones de diferentes genotipos también. A continuación, describimos mostrar nuestro protocolo de electroporación de embriones de rata E15, pero en el útero de la electroporación se realiza rutinariamente en las ratas entre las edades de E13 y E18. Mientras que las primeras etapas de la electroporación objetivos capas profundas de la corteza, más tarde electroporations fase de destino capas más superficiales. Los animales se les administró una dosis preoperatoria de la buprenorfina (0.05 a 0.1 mg / kg) antes de la cirugía se inicia. Hay múltiples opciones para anestesiar al animal. Walantus et al. Isofuorane utiliza la inhalación, mientras que nosotros usamos la inyección intraperitoneal de ketamina (40-80 mg / kg) y xilazina (5-10 mg / kg) 1,2. Una pizca pies siempre deben ser efectuados para garantizar que los animales son completamente anestesiados y no responde. Los animales se mantienen en un cojín de calefacción durante todo el procedimiento quirúrgico. El pelaje del animal se afeita en la región de la incisión, y se lavan tres veces con etanol seguido por tres veces con el yodo. Se hace una incisión en la piel, justo lateral a la línea media, seguido por una incisión en el músculo. Los cuernos uterinos están expuestos con mucho cuidado. Son suavemente extraer de la cavidad del cuerpo con los dedos. Manténgase hidratado embriones con PBS estéril mientras están fuera de la cavidad del cuerpo. </li><li> ADN por vía intravenosa y la electroporación Al empezar la realización de estas cirugías, la parte más difícil es familiarizarse con el lugar donde tiene que inyectar el ADN de manera que usted llena los ventrículos laterales, y acostumbrarse a la profundidad que se inyecte la aguja con el fin de golpear la región correcta. Los embriones son manipulados con cuidado con los dedos para que pueda identify donde está la cabeza, y si te fijas bien podrás ver la sutura línea media. Esto sirve como un punto de referencia general que puede utilizar para determinar dónde está el ventrículo lateral se encuentra. Se inyecta el ADN a través de la pared uterina y en el ventrículo lateral. Nosotros usamos un pedal para controlar la inyección del ADN - múltiples pulsos de ADN se realizan hasta el ventrículo lateral se llena con el ADN / mezcla de colorantes. A continuación, colocar los electrodos remo a cada lado de la cabeza del embrión y el uso de otro pedal para entregar el pulso en la cabeza del embrión. La colocación de los electrodos es fundamental para determinar a qué región de la corteza es electroporación. Dado que el ADN está cargado negativamente, el ADN se desplazará hacia el electrodo positivo cuando una carga se disipa a través de las paletas. Dependiendo de la ubicación exacta de los electrodos, los diferentes subconjuntos de células se destinará. En forma rutinaria colocar el electrodo positivo cerca de las posiciones dorsal-medial a través del cerebro. Sin embargo, el laboratorio LoTurco maravillosamente demostró que si se colocan los electrodos en las regiones más ventral lateral del cerebro puede dirigirse a las células de las células de la corteza-estriado límite y éxito de la corriente lateral cortical 6. Cada embrión puede ser electroporación, y diferentes combinaciones de construcciones de ADN se pueden utilizar en cada embrión. </li><li> Sutura y el cuidado post-operatorio Después de la electroporación de todos los embriones, los cuernos uterinos son cuidadosamente dentro de la cavidad del cuerpo, y tanto la capa muscular y la piel se suturan. La técnica para esto es descrito en Walantus et al 1,2. Los animales son controlados continuamente hasta que se recupere de la anestesia y el analgésico buprenorfina (0,05 a 0,1 mg / kg) se administra cada 8-12 horas. </li></ol> 2. Cultivo electroporated neuronas corticales <ol><li> La recolección y la disección de los cerebros de electroporación región electroporated Para los análisis in vivo después de la electroporación en el útero, los animales pueden ser cosechadas en cualquier punto de tiempo de 24 horas después de la electroporación a poco después de nacer a la edad adulta. Sin embargo, cuando las neuronas de cultivo primario que cosecha 24 horas después de la electroporación en E16. En este momento, los embriones electroporated expresan niveles detectables de BPA. Los animales son sacrificados con inhalación de dióxido de carbono y la decapitación rápida. Los embriones son diseccionados en el útero y se coloca en HBSS con cationes bivalentes, no perder de vista que los embriones fueron electroporated con la que los plásmidos de ADN. Es muy importante utilizar HBSS filtrado, tubos estériles y platos y herramientas en autoclave para la disección. Las cortezas son diseccionados y las meninges retirado usando un microscopio en una campana. Estas cortezas continuación se observan en un microscopio con la capacidad de visualizar las buenas prácticas agrarias. GFP positivos regiones de la corteza se identifican, y ue un par de tijeras para cortar Vanna las regiones GFP positivos de la corteza. Estas piezas se colocan en HBSS sin cationes bivalentes en un tubo cónico de 15 ml. </li><li> Disociación y las planchas de las neuronas Una vez que todas las regiones GFP positivos son disecados, HBSS se sustituye con tripsina 0,25% y se incubaron a 37 grados durante 5 minutos. Tripsina se elimina, sustituido por los medios de comunicación de chapado (DMEM + 5% de SFB + Penn / estreptomicina glutamina +), y se trituró 5-7 veces a disociar las células. Volúmenes utilizados dependen de la cantidad presente de los tejidos. Las células disociadas se está chapada en CC2 cámara de diapositivas recubiertos. Durante dos portaobjetos de cámara, que la placa de 200,000-350,000 células por la cámara en un volumen de 1,5 ml de los medios de recubrimiento. Después de 4 horas, los medios de recubrimiento se aspira y se sustituye con 1,5 ml de medio de cultivo neuronal (Neurobasal los medios de comunicación + B27 + suplemento glutamax + gentamicina) por cámara. </li></ol> 3. Analizar consecuencia del proceso neuronal <ol><li> Fijación y tinción culturas A fin de medir a corto plazo effecs de la manipulación genética de estas células, las neuronas de la cosecha principal, después de tres días in vitro. Si las neuronas primarias se cultivaron más tiempo para análisis adicionales, la mitad de los medios de comunicación debe ser reemplazado cada tres días. Para la fijación de las culturas, que aspiran los medios de comunicación de las cámaras y la solución de las neuronas en el 4% paraformaldhyde durante 15 minutos. Después de la fijación, se lavan las células dos veces en PBS y luego poner en solución de bloqueo (2% de suero de burro con 0,1% Triton X-100 en PBS) durante una hora. Células se incubaron en anticuerpo primario durante 1 hora. Para el análisis de un proceso neuronal consecuencia, utilizamos anticuerpos anti-beta tubululin para identificar las neuronas - beta tubulina inmunotinción II etiquetas neruonal célula del cuerpo, las dendritas y los axones. Las células se lavan tres veces en PBS durante cinco minutos, y luego incubadas en Cy3 anti-ratón durante 1 hora, seguido por otros tres lavados con PBS, contratinción núcleos con DAPI y montaje. </li><li> Medición de neurite longitud Imágenes de la GFP positivas las neuronas, la tubulina beta-III positivo se adquieren en un Axioskop Zeiss con un sistema de cámara MC100. Varias variables puede ser examinada en estas células GFP positivos, incluida la duración de todas las neuritas, la longitud de las más largas neuritas, la ramificación de las neuritas, el tamaño del soma celular, etc Hemos utilizado este método para analizar el proceso neuronal a consecuencia derribar o sobreexpresión de genes relacionados con el neurodesarrollo y la neurodegeneración, con un enfoque en la longitud proceso neuronal. A fin de medir consecuencia los procesos neuronales, usamos Axiovision LE 4.4 del software (de Zeiss). Dentro de este software, hay una opción para seleccionar el &quot;perfil&quot; de la herramienta. Con esta herramienta, usted puede usar el ratón del ordenador para rastrear la longitud de cada proceso neuronal. Es fundamental definir el objetivo que está utilizando con el fin de obtener una medida exacta de sus neuritas. Para estos análisis, que suelen utilizar un objetivo de 20x. </li></ol> 4. Resultados representante Hemos encontrado que Sprague Dawley tamaño de la camada oscila entre 6 y 14 embriones. Por lo general, electroporar todos los embriones. Cada embrión puede ser electroporación con una combinación diferente de AND. Sin embargo, por lo general electroporar por lo menos cuatro cerebros con la misma condición y la piscina antes de disociar estos cerebros y de las planchas. Hemos encontrado que con esta técnica un 75% de los cerebros de electroporación se dirigen a la región deseada de la corteza, ya sea corteza dorsal lateral medial o ventral (Figura 1). Además, hemos encontrado electroporations temprano en E13-14 neuronas profunda capa de destino, tales como la capa de neuronas positivas Tbr1 VI, mientras que más tarde electroporations objetivo positivo Ctip2, TBR1 células negativas V capa, y más tarde objetivo electroporations Brn2 positivo capa II / III células. Una excelente descripción de los diferentes marcadores y la explicación de la especificación de subtipo neuronal en la corteza de encontrar un artículo de Moleneaux et al 7. La figura 2 muestra las secciones coronal del cerebro de electroporación, ya sea en el día embrionario 15.5 o 17.5 y cosechadas en el día postnatal 5. Muestra en rojo es la inmunotinción para Oct6. Puede inmunotinción para marcadores de la cultura para confirmar lo que las poblaciones de células de capa que está orientado. Hemos encontrado que se puede esperar a centrarse en la población de células misma capa de células en cada embrión de la misma camada (en otras palabras, la selección depende del punto de tiempo en lugar de embriones en otras variantes técnicas). En la cultura, el porcentaje de células que son positivas las buenas prácticas agrarias pueden variar ampliamente dependiendo de lo conservadora que es cuando la disección de la región GFP positivos (Figura 3). Sin embargo, incluso cuando son muy conservadores y diseccionar sólo el parche GFP positivos de las células, el porcentaje más alto que se observa es del 5-10% - a pesar de que son la disección de la región de la corteza que se electroporación, las células en una sola capa ser atacados. Este effciency transfección de baja es de gran ayuda en la identificación de los procesos de pertenecer a la célula electoporated que se está analizando. Las células de placas en esta densidad superior contribuye a que heathier culturas, sin embargo, es difícil discernir qué proceso pertenece a cada cuerpo celular en las células GFP negativas (Figura 3). Si tiene problemas para ver todos los procesos finos de las células por electroporación, puede aumentar la concentración de ADN que son las buenas prácticas agrarias electroporating para aumentar la expresión de la GFP, o puede inmunotinción de las células disociadas utilizando un anticuerpo anti-GFP (de Invitrogen ) junto con un anticuerpo Cy2 secundaria. <img src="/files/ftp_upload/2103/2103fig1.jpg" alt="Figura 1" /> Figura 1. E15.5 ratas Sprague-Dawley fueron electroporated con el plásmido GFP y se cosecha tres días después. Sobre la base de la colocación de los electrodos, las diferentes regiones de la corteza se destinará. AF muestran la fluorescencia de GFP en el cerebro de todo. <img src="/files/ftp_upload/2103/2103fig2.jpg" alt="Figura 2" /> Figura 2. E15.5 (A) o E17.5 (B) ratas Sprague-Dawley fueron electroporación con el plásmido GFP y la cosecha en el día postnatal 5. Los cerebros fueron fijados, seccionados coronalmente con una vibratome (100 secciones micras), y immunostained para Oct6 (rojo). A y B muestran imágenes de confocal de las secciones de inmuno-. <img src="/files/ftp_upload/2103/2103fig3.jpg" alt="Figura 3" /> Figura 3. E15.5 ratas Sprague-Dawley fueron electroporated con el plásmido GFP. 24 horas después de la electroporación, los cerebros fueron cosechadas y las buenas prácticas agrarias-positivas, las regiones electroporación fueron disecados y disociar, como se describe en el vídeo. Después de 3 días in vitro, las células fueron fijadas y immunostained para BIII-tubulina (rojo) y la tinción de los núcleos con DAPI (azul) (A, C). La longitud de las más largas neuritas se midió utilizando Axiovision software(B, D).

<ol>
	<li>Walantus, W., Castaneda, D., Elias, L., Kriegstein, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+utero+intraventricular+injection+and+electroporation+of+E15+mouse+embryos.">In utero intraventricular injection and electroporation of E15 mouse embryos.</a> J Vis Exp. , (2007).</li><li>Walantus, W., Elias, L., Kriegstein, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+utero+intraventricular+injection+and+electroporation+of+E16+rat+embryos.">In utero intraventricular injection and electroporation of E16 rat embryos.</a> J Vis Exp. , (2007).</li><li>Takahashi, M., Sato, K., Nomura, T., Osumi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manipulating+gene+expressions+by+electroporation+in+the+developing+brain+of+mammalian+embryos.">Manipulating gene expressions by electroporation in the developing brain of mammalian embryos.</a> Differentiation. 70 (4-5), 155-1562 (2002).</li><li>Bai, J., Ramos, R. L., Ackman, J. B., Thomas, A. M., Lee, R. V., LoTurco, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAi+reveals+doublecortin+is+required+for+radial+migration+in+rat+neocortex.">RNAi reveals doublecortin is required for radial migration in rat neocortex.</a> Nat Neurosci. , 1277-1283 (2003).</li><li>Okada, A., Lansford, R., Weimann, J. M., Fraser, S. E., McConnell, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+cells+in+the+developing+nervous+system+with+retrovirus+expressing+modified+green+fluorescent+protein.">Imaging cells in the developing nervous system with retrovirus expressing modified green fluorescent protein.</a> Exp Neurol. 156 (2), 394-406 (1999).</li><li>Bai, J., Ramos, R. L., Paramasivam, M., Siddiqi, F., Ackman, J. B., LoTurco, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+DCX+and+LIS1+in+migration+through+the+lateral+cortical+stream+of+developing+forebrain.">The role of DCX and LIS1 in migration through the lateral cortical stream of developing forebrain.</a> Dev Neurosci. , 144-156 (2008).</li><li>Molyneaux, B. J., Arlotta, P., Menezes, J. R., Macklis, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+subtype+specification+in+the+cerebral+cortex.">Neuronal subtype specification in the cerebral cortex.</a> Nat Rev Neurosci. , 427-437 (2007).</li><li>Young-Pearse, T. L., Bai, J., Chang, R., Zheng, J. B., Loturco, J. J., Selkoe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Critical+Function+for+-Amyloid+Precursor+Protein+in+Neuronal+Migration+Revealed+by+In+Utero+RNA+Interference.">A Critical Function for -Amyloid Precursor Protein in Neuronal Migration Revealed by In Utero RNA Interference.</a> J Neurosci. 27, 14459-14469 (2007).</li><li>Young-Pearse, T. L., Chen, A. C., Chang, R., Marquez, C., Selkoe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secreted+APP+regulates+the+function+of+full-length+APP+in+neurite+outgrowth+through+interaction+with+integrin+beta1.">Secreted APP regulates the function of full-length APP in neurite outgrowth through interaction with integrin beta1.</a> Neural Develop. 3, 15-15 (2008).</li></ol>

En el estudio in vitro de cultivos neuronales primarios permiten análisis cuantitativos de crecimiento de las neuritas. Con el fin de estudiar cómo las alteraciones genéticas afectan a consecuencia proceso neuronal, las construcciones del shRNA o misexpression se pueden introducir en las neuronas primarias a través de la transfección químicos o transducción viral. Sin embargo, con células de la corteza primaria, un grupo heterogéneo de tipos de células (neuronas glutamatérgicas de diferentes...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Cortical Neuron Preparation</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Dissection Media:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>10X Hanks' Balanced Salt Solution (HBSS) (Ca+2 /Mg +2 free)</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>14185-052</td>
<td>&nbsp;</td>
<tr>
<td>10X Hanks' Balanced Salt Solution (HBSS) (with Ca+2 /Mg +2 )</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>14065-056</td>
<td>&nbsp;</td>
<tr>
<td>1M HEPES pH 7.4</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>15630-080</td>
<td>&nbsp;</td>
<tr>
<td>Dishes and Vials:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>100 x 15 mm Petri Dishes</td>
<td>
</td>
<td>Fisherbrand</td>
<td>08-757-12</td>
<td>&nbsp;</td>
<tr>
<td>60 x 15 mm Petri Dishes</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>351007</td>
<td>&nbsp;</td>
<tr>
<td>15 mL conical vial</td>
<td>&nbsp;</td>
<td>Sarstedt</td>
<td>62-547-205</td>
<td>&nbsp;</td>
<tr>
<td>50 mL conical vial</td>
<td>&nbsp;</td>
<td>Sarstedt</td>
<td>62-554-205</td>
<td>&nbsp;</td>
<tr>
<td>Dissection Tools:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Scissors </td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>91402-12</td>
<td>&nbsp;</td>
<tr>
<td>Standard Forceps</td>
<td>
</td>
<td>Fine Science Tools</td>
<td>11000-12</td>
<td>&nbsp;</td>
<tr>
<td>Curved Forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11273-20</td>
<td>&nbsp;</td>
<tr>
<td>Fine Forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11255-20</td>
<td>&nbsp;</td>
<tr>
<td>Vannas spring scissors </td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>15000-00</td>
<td>&nbsp;</td>
<tr>
<td>Miscellaneous:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>.25% Trypsin-EDTA</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>25200</td>
<td>&nbsp;</td>
<tr>
<td>Reichert Bright-Line Hemacytometer</td>
<td>&nbsp;</td>
<td>Hausser Scientific</td>
<td>1490</td>
<td>&nbsp;</td>
<tr>
<td>Hand-Held Tally Counter</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>Z169021</td>
<td>&nbsp;</td>
<tr>
<td>Plating Medium:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Dulbecco's Modified Eagle Medium (D-MEM)</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>11960-051</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F4135</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-Streptomycin</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>L-glutamine</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>25030</td>
<td>&nbsp;</td>
<tr>
<td>Growth Medium:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>NEUROBASAL Medium </td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>21103-049</td>
<td>&nbsp;</td>
<tr>
<td>B-27 Serum-Free Supplement </td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>17504-044</td>
<td>&nbsp;</td>
<tr>
<td>GlutaMAX -I Supplement</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>35050-061</td>
<td>&nbsp;</td>
<tr>
<td>Gentamicin Reagent Solution </td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>15750-060</td>
<td>&nbsp;</td>
<tr>
<td>Immunostaining:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fixative, Washes, and Blocking Buffer:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Paraformaldehyde</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P6148</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate Buffered Saline</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P4417</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T9284</td>
<td>&nbsp;</td>
<tr>
<td>Donkey Serum</td>
<td>&nbsp;</td>
<td>Jackson Immuno</td>
<td>017-000-121 </td>
<td>&nbsp;</td>
<tr>
<td>Antibodies:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>beta-III tubulin antibody</td>
<td>&nbsp;</td>
<td>Chemicon</td>
<td>MAB1637</td>
<td>&nbsp;</td>
<tr>
<td>MAP2 antibody</td>
<td>&nbsp;</td>
<td>Chemicon</td>
<td>AB15452</td>
<td>&nbsp;</td>
<tr>
<td>Donkey Cy3 anti-mouse</td>
<td>&nbsp;</td>
<td>Jackson Immuno</td>
<td>715-166-151</td>
<td>&nbsp;</td>
<tr>
<td>Donkey Cy2 anti-chicken</td>
<td>&nbsp;</td>
<td>Jackson Immuno</td>
<td>703-226-155 </td>
<td>&nbsp;</td>
<tr>
<td>DAPI</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>D3571</td>
<td>&nbsp;</td>
<tr>
<td>Slide Preparation:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>CC2 Coated Two-Chamber Slides </td>
<td>&nbsp;</td>
<td>Lab-Tek</td>
<td>154852</td>
<td>&nbsp;</td>
<tr>
<td>Fluorescent Mounting Media</td>
<td>&nbsp;</td>
<td>KPL</td>
<td>71-00-16</td>
<td>&nbsp;</td>
<tr>
<td>24 x 60 mm Micro Cover Glasses</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>48393-106 </td>
<td>&nbsp;</td>
<tr>
<td>Clear nail polish</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>72180</td>
<td>&nbsp;</td>
<tr>
<td>Electroporation:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Ketamine</td>
<td>&nbsp;</td>
<td>Henry Schein</td>
<td>995-2949</td>
<td>&nbsp;</td>
<tr>
<td>Xylazine</td>
<td>&nbsp;</td>
<td>Henry Schein</td>
<td>4015809TV</td>
<td>&nbsp;</td>
<tr>
<td>buprenorphine</td>
<td>&nbsp;</td>
<td>Henry Schein</td>
<td>1118217</td>
<td>&nbsp;</td>
<tr>
<td>Picospritzer III</td>
<td>&nbsp;</td>
<td>Parker</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>BTX square wave electroporator</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BTXECM830</td>
<td>&nbsp;</td>
<tr>
<td>Tweezertrodes, 7 mm, platinum</td>
<td>&nbsp;</td>
<td>Harvard Apparatus</td>
<td>450488</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

in utero electroporation followed by primary neuronal culture for studying gene function in subset of cortical neurons

Estudio in vitro de cultivos primarios de neuronas permite un análisis cuantitativo de crecimiento de las neuritas. Con el fin de estudiar cómo las alteraciones genéticas afectan a consecuencia proceso neuronal, shRNA o construcciones de ADNc se pueden introducir en las neuronas primarias a través de la transfección químicos o transducción viral. Sin embargo, con células de la corteza primaria, un grupo heterogéneo de tipos de células (neuronas glutamatérgicas de diferentes capas, las neuronas inhibidoras, las células gliales) se transfectaron usando estos métodos. El uso de la electroporación en el útero para introducir construcciones de ADN en la corteza roedores embrionarias permite ciertos subconjuntos de células para ser específicos: mientras que la electroporación de los primeros objetivos de embriones corteza capas profundas de la corteza, la electroporación a finales de los puntos de tiempo embrionarias objetivos de las capas más superficiales. Además, la colocación de electrodos diferenciales a través de las cabezas de los resultados de embriones individuales en la focalización de las regiones dorsal-ventral medial frente al lateral de la corteza. Después de la electroporación, las células transfectadas se disecaron, disociados, y se colocaron in vitro para el análisis cuantitativo de crecimiento de las neuritas. Aquí, ofrecemos un método paso a paso para medir cuantitativamente consecuencia proceso neuronal en los subconjuntos de células corticales. El protocolo básico para la electroporación en el útero ha sido descrito en detalle en dos artículos JoVe otros del laboratorio Kriegstein 1, 2. Vamos a ofrecer una visión general de nuestro protocolo de electroporación en el útero, se centra en los detalles más importantes, seguido de una descripción de nuestro protocolo que se aplica en la electroporación in utero al estudio de la función de genes en consecuencia el proceso neuronal.

Watch this Scientific Journal Video about In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons at JoVE.com

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

En la electroporación in utero es un método valioso para la transfección de células progenitoras neuronales in vivo. Dependiendo de la colocación de los electrodos y el punto de tiempo en el desarrollo de la electroporación, ciertos subconjuntos de células corticales pueden ser atacadas. Células afectadas puede ser analizada en vivo o in vitro para los efectos de la alteración genética.

En el útero de electroporación seguido por cultivos neuronales primarias para estudiar la función génica en el subconjunto de neuronas corticales

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

in-utero-electroporation-followed-primary-neuronal-culture-for

Research

JoVE Journal

Neuroscience

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

17.6K vistas.  Brigham and Woman's Hospital and Harvard Medical School. En la electroporación in utero es un método valioso para la transfección de células progenitoras neuronales in vivo. Dependiendo de la colocación de los electrodos y el punto de tiempo en el desarrollo de la electroporación, ciertos subconjuntos de células corticales pueden ser atacadas. Células afectadas puede ser analizada en vivo o in vitro para los efectos de la alteración genética.

Video: In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Nucleofection y cultivos primarios de neuronas del hipocampo de embriones de ratón y corticales

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

Investigación

Neurociencias

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental pathways. Several powerful and well established transgenic technologies are available to manipulate gene expression levels in mouse, allowing for the generation of both loss- and gain-of-function models. However, the generation of mouse transgenics is both costly and time consuming. Alternative methods of gene manipulation have therefore been widely sought. In utero electroporation is a method of gene delivery into live mouse embryos1,2 that we have successfully adapted3,4. It is largely based on the success of in ovo electroporation technologies that are commonly used in chick5. Briefly, DNA is injected into the open ventricles of the developing brain and the application of an electrical current causes the formation of transient pores in cell membranes, allowing for the uptake of DNA into the cell. In our hands, embryos can be efficiently electroporated as early as embryonic day (E) 11.5, while the targeting of younger embryos would require an ultrasound-guided microinjection protocol, as previously described6. Conversely, E15.5 is the latest stage we can easily electroporate, due to the onset of parietal and frontal bone differentiation, which hampers microinjection into the brain. In contrast, the retina is accessible through the end of embryogenesis. Embryos can be collected at any time point throughout the embryonic or early postnatal period. Injection of a reporter construct facilitates the identification of transfected cells.
To date, in utero electroporation has been most widely used for the analysis of neocortical development1,2,3,4. More recent studies have targeted the embryonic retina7,8,9 and thalamus10,11,12. Here, we present a modified in utero electroporation protocol that can be easily adapted to target different domains of the embryonic CNS. We provide evidence that by using this technique, we can target the embryonic telencephalon, diencephalon and retina. Representative results are presented, first showing the use of this technique to introduce DNA expression constructs into the lateral ventricles, allowing us to monitor progenitor maturation, differentiation and migration in the embryonic telencephalon. We also show that this technique can be used to target DNA to the diencephalic territories surrounding the 3rd ventricle, allowing the migratory routes of differentiating neurons into diencephalic nuclei to be monitored. Finally, we show that the use of micromanipulators allows us to accurately introduce DNA constructs into small target areas, including the subretinal space, allowing us to analyse the effects of manipulating gene expression on retinal development.

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

In utero electroporation allows for rapid gene delivery in a spatially- and temporally-controlled manner in the developing central nervous system (CNS). Here we describe a highly adaptable in utero electroporation protocol that can be used to deliver expression constructs into multiple embryonic CNS domains, including the telencephalon, diencephalon and retina.

Entrega génica eficaz en los territorios del SNC múltiples utilizando In Utero La electroporación

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental ...

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

Del ratón en el útero de electroporación: Transefection controlada Gene espacio-temporal

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

Bilaminar Co-cultivo de neuronas de rata primaria cortical y la glía

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain dopaminergic neurons is implicated in Parkinson's disease, Schizophrenia, depression, and dementia1-5. Thus, studying the regulation of midbrain dopaminergic neuron differentiation could not only provide important insight into mechanisms regulating midbrain development and neural progenitor fate specification, but also help develop new therapeutic strategies for treating a variety of human neurological disorders.
Dopaminergic neurons differentiate from neural progenitors lining the ventricular zone of embryonic ventral midbrain. The development of neural progenitors is controlled by gene expression programs6,7. Here we report techniques utilizing electroporation to express genes specifically in the midbrain of Hamburger Hamilton (HH) stage 11 (thirteen somites, 42 hours) chick embryos8,9. The external development of chick embryos allows for convenient experimental manipulations at specific embryonic stages, with the effects determined at later developmental time points10-13. Chick embryonic neural tubes earlier than HH stage 13 (nineteen somites, 48 hours) consist of multipotent neural progenitors that are capable of differentiating into distinct cell types of the nervous system. The pCAG vector, which contains both a CMV promoter and a chick &beta;-actin enhancer, allows for robust expression of Flag or other epitope-tagged constructs in embryonic chick neural tubes14. In this report, we emphasize special measures to achieve regionally restricted gene expression in embryonic midbrain dopaminergic neuron progenitors, including how to inject DNA constructs specifically into the embryonic midbrain region and how to pinpoint electroporation with small custom-made electrodes. Analyzing chick midbrain at later stages provides an excellent in vivo system for plasmid vector-mediated gain-of-function and loss-of-function studies of midbrain development. Modification of the experimental system may extend the assay to other parts of the nervous system for performing fate mapping analysis and for investigating the regulation of gene expression.

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

To assess the function and the regulation of genes during the development of midbrain dopaminergic neurons, we describe a method that involves in ovo electroporation of plasmid DNA constructs into embryonic chick ventral midbrain dopaminergic neuron progenitors. This technique can be used to achieve efficient expression of genes of interest to study different aspects of midbrain development and dopaminergic neuron differentiation.

En electroporación in ovo en el mesencéfalo polluelo para estudiar la función génica en el desarrollo neuronal dopaminérgica

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain ...

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the use of IUE for investigating behavior or neuropathology in the adult brain has been limited by insufficient methods for monitoring of IUE transfection success by non-invasive techniques in postnatal animals. 
 For the present study, E16 rats were used for IUE. After intraventricular injection of the nucleic acids into the embryos, positioning of the tweezer electrodes was critical for targeting either the developing cortex or the hippocampus. 
 Ventricular co-injection and electroporation of a luciferase gene allowed monitoring of the transfected cells postnatally after intraperitoneal luciferin injection in the anesthetized live P7 pup by in vivo bioluminescence, using an IVIS Spectrum device with 3D quantification software. 
 Area definition by bioluminescence could clearly differentiate between cortical and hippocampal electroporations and detect a signal longitudinally over time up to 5 weeks after birth. This imaging technique allowed us to select pups with a sufficient number of transfected cells assumed necessary for triggering biological effects and, subsequently, to perform behavioral investigations at 3 month of age. As an example, this study demonstrates that IUE with the human full length DISC1 gene into the rat cortex led to amphetamine hypersensitivity. Co-transfected GFP could be detected in neurons by post mortem fluorescence microscopy in cryosections indicating gene expression present at &ge;6 months after birth.
 We conclude that postnatal bioluminescence imaging allows evaluating the success of transient transfections with IUE in rats. Investigations on the influence of topical gene manipulations during neurodevelopment on the adult brain and its connectivity are greatly facilitated. For many scientific questions, this technique can supplement or even replace the use of transgenic rats and provide a novel technology for behavioral neuroscience.

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

Genes can be manipulated during development of the cortex or the hippocampus of the rat via in utero electroporation (IUE) at E16, to enable fast and targeted modifications in neuronal connectivity for later studies of behavior or neuropathology in adult animals. Postnatal in vivo imaging for control of IUE success is performed by bioluminescence of activating co-transfected luciferase.

Generación de ratas transgénicas por vía tópica<em&gt; En el útero</em&gt; La electroporación y<em&gt; En vivo</em&gt; Detección bioluminiscencia

 In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the ...

Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis of primary cultures of neurons. We describe a set of procedures for: labeling newborn mice to be genotyped, rapid genotyping, and establishing low-density cultures of brain neurons from these mice. Individual mice are labeled by tattooing, which allows for long-term identification lasting into adulthood. Genotyping by the described protocol is fast and efficient, and allows for automated extraction of nucleic acid with good reliability. This is useful under circumstances where sufficient time for conventional genotyping is not available, e.g., in mice that suffer from neonatal lethality. Primary neuronal cultures are generated at low density, which enables imaging experiments at high spatial resolution. This culture method requires the preparation of glial feeder layers prior to neuronal plating. The protocol is applied in its entirety to a mouse model of the movement disorder DYT1 dystonia (&#916;E-torsinA knock-in mice), and neuronal cultures are prepared from the hippocampus, cerebral cortex and striatum of these mice. This protocol can be applied to mice with other genetic mutations, as well as to animals of other species. Furthermore, individual components of the protocol can be used for isolated sub-projects. Thus this protocol will have wide applications, not only in neuroscience but also in other fields of biological and medical sciences.

We describe procedures for labeling and genotyping newborn mice and generating primary neuronal cultures from them. The genotyping is rapid, efficient and reliable, and allows for automated nucleic-acid extraction. This is especially useful for neonatally lethal mice and their cultures that require prior completion of genotyping.

Rápido Genotipado de animales seguidas del establecimiento de cultivos primarios de neuronas cerebrales

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis ...

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, their distinct dendritic morphologies, their specific patterns of axonal connectivity, and their selective firing responses. The molecular and cellular mechanisms responsible for these aspects of differentiation during development are still poorly understood.
Here, we describe combined protocols for labeling and characterizing the structural connectivity and excitability of cortical neurons. Modification of the in utero electroporation (IUE) protocol allows the labeling of a sparse population of neurons. This, in turn, enables the identification and tracking of the dendrites and axons of individual neurons, the precise characterization of the laminar location of axonal projections, and morphometric analysis. IUE can also be used to investigate changes in the excitability of wild-type (WT) or genetically modified neurons by combining it with whole-cell recording from acute slices of electroporated brains. These two techniques contribute to a better understanding of the coupling of structural and functional connectivity and of the molecular mechanisms controlling neuronal diversity during development. These developmental processes have important implications on axonal wiring, the functional diversity of neurons, and the biology of cognitive disorders.

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

This manuscript provides protocols that use in utero electroporation (IUE) to describe the structural connectivity of neurons at the single-cell level and the excitability of fluorescently labeled neurons. Histology is used to characterize dendritic and axonal projections. Whole-cell recording in acute slices is used to investigate excitability.

La electroporación en el útero métodos para estudiar la excitabilidad de las subpoblaciones neuronales y Conectividad Una sola célula

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

Modelado de la muerte Neuronal y la degeneración en las neuronas del gránulo cerebeloso primario de ratón

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It has helped to describe the different steps of radial migration and characterize the molecular mechanisms controlling this process. To directly and dynamically analyze migrating neurons they have to be traced over time. This protocol describes a workflow that combines in utero electroporation with organotypic slice culture and time-lapse confocal imaging, which allows for a direct examination and dynamic analysis of radially migrating cortical neurons. Furthermore, detailed characterization of migrating neurons, such as migration speed, speed profiles, as well as radial orientation changes, is possible. The method can easily be adapted to perform functional analyses of genes of interest in radially migrating cortical neurons by loss and gain of function as well as rescue experiments. Time-lapse imaging of migrating neurons is a state-of-the-art technique that once established is a potent tool to study the development of the cerebral cortex in mouse models of neuronal migration disorders.

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

This protocol provides instructions for direct observation of radially migrating cortical neurons. In utero electroporation, organotypic slice culture, and time-lapse confocal imaging are combined to directly and dynamically study the effects of overexpression or downregulation of genes of interest in migrating neurons and to analyze their differentiation during development.

Time-lapse Imágenes confocales de las neuronas migratorias en la cultura organotípica en rebanadas de cerebro de ratón embrionario<em&gt; En Utero</em&gt; Electroporación

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It ...